Method for treating cancer

ABSTRACT

The present disclosure relates to pharmaceutical compositions comprising inhibitor(s) of human histone methyltransferase EZH2, and methods of cancer therapy using the EZH2 inhibitor(s).

RELATED APPLICATIONS

This application is continuation of U.S. application Ser. No.15/754,684, filed on Feb. 23, 2018, which is a U.S. National Phaseapplication, filed under 35 U.S.C. § 371, of International ApplicationNo. PCT/US2016/048401, filed on Aug. 24, 2016, which claims priority to,and the benefit of U.S. Provisional Application No. 62/209,304 filedAug. 24, 2015, the entire contents of each of which are incorporatedherein by reference in their entireties.

BACKGROUND OF THE DISCLOSURE

EZH2, a histone methyltransferase, has been associated with variouskinds of cancers. Specifically, mutations and and/or overactivity ofEZH2 are found in a range of cancers, such as lymphomas, leukemias andbreast cancer. There is an ongoing need for new agents as EZH2inhibitors for use in anticancer treatment.

SUMMARY OF THE DISCLOSURE

In one aspect, the present disclosure features a method for thetreatment or prevention of an INI1-negative tumor. The method comprisesadministering a therapeutically effective amount of an EZH2 inhibitor toa subject in need thereof.

The method can include one or more of the following features.

In one embodiment, the INI1-negative tumor is rhabdoid tumor of thekidney (RTK).

In one embodiment, the INI1-negative tumor is atypical teratoid/rhabdoidtumor (ATRT).

In one embodiment, the INI1-negative tumor is epithelioid malignantperipheral nerve sheath tumor.

In one embodiment, the INI1-negative tumor is myoepithelial carcinoma.

In one embodiment, the INI1-negative tumor is renal medullary carcinoma.

In one embodiment, the EZH2 inhibitor is administered orally.

In one embodiment, the subject is a human being.

In one embodiment, the subject is younger than 18 years.

In one embodiment, the EZH2 inhibitor is Compound (A), having thefollowing formula

or a pharmaceutically acceptable salt thereof.

As used herein, the expressions “Compound (A),” “tazemetostat,”“EPZ-6438,” and “EPZ-6438” all refer to the same Compound (A) and can beused interchangeably.

In one embodiment, the EZH2 inhibitor is administered to the subject ata dose of about 100 mg to about 3200 mg daily.

In one embodiment, the EZH2 inhibitor is administered to the subject ata dose of about 100 mg BID to about 1600 mg BID.

In one embodiment, the EZH2 inhibitor is administered to the subject ata dose of about 100 mg BID, 200 mg BID, 400 mg BID, 800 mg BID, or about1600 mg BID.

In one embodiment, the EZH2 inhibitor is administered to the subject ata dose of 800 mg BID.

In one embodiment, the EZH2 inhibitor is selected from the groupconsisting of:

and pharmaceutically acceptable salts thereof.

In one embodiment, the EZH2 inhibitor is:

or a pharmaceutically acceptable salt thereof.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents and other references mentioned herein areincorporated by reference. The references cited herein are not admittedto be prior art to the claimed disclosure. In the case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods and examples are illustrative only and are notintended to be limiting.

Any of the above aspects and embodiments can be combined with any otheraspect or embodiment.

Other features and advantages of the disclosure will be apparent fromthe following detailed description and claims.

DETAILED DESCRIPTION OF THE DISCLOSURE

Histone methyltransferases (HMTs) play a crucial role in the regulationof gene expression. In particular, HMTs are involved in the regulationof cellular division and of cellular differentiation. HMTs mediate themethylation of histones associated with particular genes. Depending onthe amino acid residues that are methylated, the methylation event caneither signal a silencing event or an activation event for theassociated gene. Examples of a silencing mark include the trimethylationof H3K27; whereas, trimethylation of H3K4 results in a gene activatingsignal. Many cell cycle check point regulators and tumor suppressorgenes exist in a “bivalent” state, wherein these contain both activatinghistone modifications (e.g. H3K27me3) and suppressing histonemodifications (e.g. H3K4me3). Genes in a bivalent state are poised toundergo either activation or suppression depending on external factors.EZH2 regulates bivalent genes involved in B-cell differentiation andmaturation, including CDKN1, PRDM1, and IRF4.

EZH2 is a histone methyltransferase that is the catalytic subunit of thePRC2 complex which catalyzes the mono- through tri-methylation of lysine27 on histone H3 (H3-K27). Histone H3-K27 trimethylation is a mechanismfor suppressing transcription of specific genes that are proximal to thesite ofhistone modification. This trimethylation is known to be a cancermarker with altered expression in cancer, such as prostate cancer (see,e.g., U.S. Patent Application Publication No. 2003/0175736; incorporatedherein by reference in its entirety). Other studies provided evidencefor a functional link between dysregulated EZH2 expression,transcriptional repression, and neoplastic transformation. Varambally etal. (2002) Nature 419(6907):624-9 Kleer et al. (2003) Proc Natl Acad SciUSA 100(20):11606-11.

EZH2 methylation activity plays an important role in the regulation andactivation of germinal center B-cells. EZH2 protein levels increasefollowing the activation of B-cells. Following activation, B-cells takeresidence in the germinal center of lymphoid organs, wherein somatichypermutation occurs, a process associated with the repression ofanti-apoptotic genes and check point regulators. EZH2 methylating eventstarget genes that are involved in B-cell proliferation, differentiationand maturation, including CDKN1A (role in cellular proliferation), PRDM1(role in B-cell differentiation) and IRF4 (role in B-celldifferentiation).

Genetic alterations within the EZH2 gene are associated with alteredhistone methylation patterns. For example, certain point mutations inEZH2 are associated with altered methylation of H3K4 in DLBCL;furthermore, chromosomal translocation and fusion, SSX:SS18, isassociated with altered H3K27 methylation in synovial sarcoma. EZH2mutations leading to the conversion of amino acid Y641 (equivalent toY646, catalytic domain), to either F, N, H, S or C results inhypertrimethylation of H3K27 and drives lymphomagenesis. Additionalgenetic alterations that affect the methylation of H3K27 include EZH2SET-domain mutations, overexpression of EZH2, overexpression of otherPRC2 subunits, loss of function mutations of histone acetyl transferases(HATs), and loss of function of MLL2. Cells that are heterozygous forEZH2 Y646 mutations result in hypertrimethylation of H3K27 relative tocells that are homozygous wild-type (WT) for the EZH2 protein, or tocells that are homozygous for the Y646 mutation.

EPZ-6438 (Compound (A)) is a small molecule inhibitor of EZH2, thecatalytic subunit of the polycomb repressive complex 2 that methylatesH3K27. Hypertrimethylation of H3K27 (H3K27Me3) appears tumorigenic invarious malignancies, including subsets of Non-Hodgkin Lymphoma (NHL)with mutant EZH2. Inhibition of H3K27Me3 with EPZ-6438 leads to killingof EZH2 mutant lymphoma cells and other EZH2 inhibitors show activity inmodels of mutant and WT EZH2 NHL. In addition, tumors with loss of INI1,a subunit of the SWI-SNF chromatin remodeling complex, appeareddependent on EZH2. EPZ-6438 was shown to induce apoptosis anddifferentiation of INI1-deleted malignant rhabdoid tumor (MRT) models invitro and in MRT xenograft-bearing mice.

This disclosure is based on, at least in part, discovery that Enhancerof Zeste Homolog 2 (EZH2) inhibitors may effectively treat cancer(s),for example cancer(s) that are characterized by aberrant H3-K27methylation.

An aspect of the present disclosure relates to a method for treating orpreventing an INI1-negative tumor. The method comprises administering atherapeutically effective amount of an EZH2 inhibitor to a subject inneed thereof. Another aspect relates to a method for treating anINI1-negative tumor. In another aspect, the present disclosure relatesto a method for treating an INI1-negative tumor comprising administeringa therapeutically effective amount of an EZH2 inhibitor to a subject inneed thereof, wherein the INI1-negative tumor is selected from rhabdoidtumor of the kidney (RTK), atypical teratoid/rhabdoid tumor (ATRT),epithelioid malignant peripheral nerve sheath tumor, myoepithelialcarcinoma, and renal medullary carcinoma.

Another aspect of the present disclosure relates to a method fortreating or preventing a rhabdoid tumor of the kidney (RTK). In anotheraspect, the present disclosure relates to a method for treating orpreventing an atypical teratoid/rhabdoid tumor (ATRT).

Another aspect of the present disclosure relates to a method fortreating or preventing an epithelioid malignant peripheral nerve sheathtumor. In another aspect, the present disclosure relates to a method fortreating or preventing a myoepithelial carcinoma. Another aspect of thepresent disclosure relates to a method for treating or preventing arenal medullary carcinoma.

In one embodiment, the EZH2 inhibitor is administered orally.

In one embodiment, the subject is a human being.

In one embodiment, the subject is younger than 18 years. In certainembodiments, in any method described herein, the subject is an adultpatient aged 18 years or older.

In certain embodiments, in any method described herein, the subject is apediatric patient aged 12 months or younger (e.g., between 3 and 12months old).

In certain embodiments, in any method described herein, the subject is asubject older than 12 months but younger than 18 years old.

In any method described herein, the subject can be a pediatric(non-adult) patient aged 3 months to 18 years.

In any of the above aspects or embodiments, the disclosure also relatesto methods for detecting levels of histone methylation, e.g., H3K27trimethylation, in a skin biopsy. Histone methylation is detected priorto initiation of treatment, while the subject is receiving treatment,and/or after treatment has concluded.

In one embodiment, the compound suitable for the methods disclosedherein is EPZ-6438 (tazemetostat):

or a pharmaceutically acceptable salt thereof.

EPZ-6438 or a pharmaceutically acceptable salt thereof, as describedherein, is potent in targeting both WT and mutant EZH2. EPZ-6438 isorally bioavailable and has high selectivity to EZH2 compared with otherhistone methyltransferases (i.e. >20,000 fold selectivity by Ki).Importantly, EPZ-6438 has target methyl mark inhibition that results inthe killing of genetically defined cancer cells in vitro. Animal modelshave also shown sustained in vivo efficacy following inhibition oftarget methyl mark. Clinical trial results described herein alsodemonstrate the safety and efficacy of EPZ-6438.

In one embodiment, EPZ-6438 or a pharmaceutically acceptable saltthereof is administered to the subject at a dose of approximately 100 mgto approximately 3200 mg daily, such as about 100 mg BID to about 1600mg BID (e.g., 100 mg BID, 200 mg BID, 400 mg BID, 800 mg BID, or 1600 mgBID), for treating a NHL. In one embodiment the dose is 800 mg BID.

In some embodiments, a compound (e.g., EZH2 inhibitor) that can be usedin any methods presented here is:

or stereoisomers thereof or pharmaceutically acceptable salts andsolvates thereof.

In certain embodiments, a compound that can be used in any methodspresented here is Compound F:

or pharmaceutically acceptable salts thereof.

In some embodiments, a compound (e.g., EZH2 inhibitor) that can be usedin any methods presented here is GSK-126 having the following formula:

stereoisomers thereof, or pharmaceutically acceptable salts or solvatesthereof.

In certain embodiments, a compound that can be used in any methodspresented here is Compound G:

or stereoisomers thereof or pharmaceutically acceptable salts andsolvates thereof.

In certain embodiments, a compound (e.g., EZH2 inhibitor) that can beused in any methods presented here is any of Compounds Ga-Gc:

or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, a compound (e.g., EZH2 inhibitor) that can beused in any methods presented here is CPI-1205 or GSK343.

Additional suitable EZH2 inhibitors will be apparent to those skilled inthe art. In some embodiments of the strategies, treatment modalities,methods, combinations, and compositions provided herein, the EZH2inhibitor is an EZH2 inhibitor described in U.S. Pat. No. 8,536,179(describing GSK-126 among other compounds and corresponding to WO2011/140324), the entire contents of each of which are incorporatedherein by reference.

In some embodiments of the strategies, treatment modalities, methods,combinations, and compositions provided herein, the EZH2 inhibitor is anEZH2 inhibitor described in PCT/US2014/015706, published as WO2014/124418, in PCT/US2013/025639, published as WO 2013/120104, and inU.S. Ser. No. 14/839,273, published as US 2015/0368229, the entirecontents of each of which are incorporated herein by reference.

In one embodiment, the compound disclosed herein is the compound itself,i.e., the free base or “naked” molecule. In another embodiment, thecompound is a salt thereof, e.g., a mono-HCl or tri-HCl salt, mono-HBror tri-HBr salt of the naked molecule.

Compounds disclosed herein that contain nitrogens can be converted toN-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds suitable for any methods disclosed herein. Thus, allshown and claimed nitrogen-containing compounds are considered, whenallowed by valency and structure, to include both the compound as shownand its N-oxide derivative (which can be designated as N→O or N⁺—O—).Furthermore, in other instances, the nitrogens in the compoundsdisclosed herein can be converted to N-hydroxy or N-alkoxy compounds.For example, N-hydroxy compounds can be prepared by oxidation of theparent amine by an oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.”

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cylcobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds disclosed herein may bedepicted as different chiral isomers or geometric isomers. It shouldalso be understood that when compounds have chiral isomeric or geometricisomeric forms, all isomeric forms are intended to be included in thescope of the disclosure, and the naming of the compounds does notexclude any isomeric forms.

Furthermore, the structures and other compounds discussed in thisdisclosure include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine. An example of keto-enol equilibria is betweenpyridin-2(1H)-ones and the corresponding pyridin-2-ols, as shown below.

It is to be understood that the compounds disclosed herein may bedepicted as different tautomers. It should also be understood that whencompounds have tautomeric forms, all tautomeric forms are intended to beincluded in the scope of the disclosure, and the naming of the compoundsdoes not exclude any tautomer form.

The compounds disclosed herein include the compounds themselves, as wellas their salts and their solvates, if applicable. A salt, for example,can be formed between an anion and a positively charged group (e.g.,amino) on an aryl- or heteroaryl-substituted benzene compound. Suitableanions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate,nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate,glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate,tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, andacetate (e.g., trifluoroacetate). The term “pharmaceutically acceptableanion” refers to an anion suitable for forming a pharmaceuticallyacceptable salt. Likewise, a salt can also be formed between a cationand a negatively charged group (e.g., carboxylate) on an aryl- orheteroaryl-substituted benzene compound. Suitable cations include sodiumion, potassium ion, magnesium ion, calcium ion, and an ammonium cationsuch as tetramethylammonium ion. The aryl- or heteroaryl-substitutedbenzene compounds also include those salts containing quaternarynitrogen atoms. In the salt form, it is understood that the ratio of thecompound to the cation or anion of the salt can be 1:1, or any rationother than 1:1, e.g., 3:1, 2:1, 1:2, or 1:3.

Additionally, the compounds disclosed herein, for example, the salts ofthe compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds disclosed herein arearyl- or heteroaryl-substituted benzene compounds.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

In certain aspects, “combination therapy” also embraces theadministration of the therapeutic agents as described above in furthercombination with other biologically active ingredients and non-drugtherapies (e.g., surgery or radiation treatment). Where the combinationtherapy further comprises a non-drug treatment, the non-drug treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andnon-drug treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

In another aspect, a composition disclosed herein, or a pharmaceuticallyacceptable salt, solvate, analog or derivative thereof, may beadministered in combination with radiation therapy. Radiation therapycan also be administered in combination with a composition disclosedherein and another chemotherapeutic agent described herein as part of amultiple agent therapy.

A “pharmaceutical composition” is a formulation containing a compound ina form suitable for administration to a subject. A compound disclosedherein and one or more other therapeutic agents described herein eachcan be formulated individually or in multiple pharmaceuticalcompositions in any combinations of the active ingredients. Accordingly,one or more administration routes can be properly elected based on thedosage form of each pharmaceutical composition. Alternatively, acompound disclosed herein and one or more other therapeutic agentsdescribed herein can be formulated as one pharmaceutical composition.

In one embodiment, the pharmaceutical composition is in bulk or in unitdosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this disclosure includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition disclosed herein is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A composition disclosed herein can be administered to a subject in manyof the well-known methods currently used for chemotherapeutic treatment.For example, for treatment of cancers, a compound disclosed herein maybe injected directly into tumors, injected into the blood stream or bodycavities or taken orally or applied through the skin with patches. Thedose chosen should be sufficient to constitute effective treatment butnot so high as to cause unacceptable side effects. The state of thedisease condition (e.g., cancer, precancer, and the like) and the healthof the patient should preferably be closely monitored during and for areasonable period after treatment.

In certain embodiments the therapeutically effective amount of eachpharmaceutical agent used in combination will be lower when used incombination in comparison to monotherapy with each agent alone. Suchlower therapeutically effective amount could afford for lower toxicityof the therapeutic regimen.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

In any method or formulation (e.g., an oral dosage form) describedherein, in one embodiment, said cancer is advanced, refractory orresistant cancer. In any method or formulation (e.g., an oral dosageform) described herein, in one embodiment, said cancer is anINI1-deficient tumor.

INI1 is a critical component of the SWI/SNF regulatory complex, achromatin remodeler that acts in opposition to EZH2. INI1-negativetumors have altered SWI/SNF function, resulting in aberrant andoncogenic EZH2 activity. This activity can be targeted by small moleculeinhibitors of EZH2 such as tazemetostat. INI1-negative tumors aregenerally aggressive and are poorly served by current treatments. Forexample, current treatment of MRT, a well-studied INI1-negative tumor,consists of surgery, chemotherapy and radiation therapy, which areassociated with limited efficacy and significant treatment-relatedmorbidity.

In any method or formulation (e.g., an oral dosage form) describedherein, in one embodiment, the subject is human.

In any method or formulation (e.g., an oral dosage form) describedherein when applicable, the cancer is a solid tumor. Examples of thesolid tumor described herein include, but are not limited to Colorectaladenocarcinoma, Cholangiocarcinoma, Pancreatic adenocarcinoma, Ewing'ssarcoma, Synovial sarcoma, Alveolar sarcoma, Alveolar soft part sarcoma,Prostatic adenocarcinoma, Rhabdoid sarcoma, Malignant Rhabdoid tumor,and Urothelial carcinoma.

In any method or formulation (e.g., an oral dosage form) describedherein when applicable, the cancer is a cancer with aberrant H3-K27methylation.

In any method or formulation (e.g., an oral dosage form) describedherein, the compound disclosed herein or a pharmaceutically acceptablesalt thereof is administered orally for at least 7, 14, 21, 28, 35, 42,47, 56, or 64 days. In certain embodiments, the administration is acontinuous administration without a drug holiday. For example, thecompound disclosed herein or a pharmaceutically acceptable salt thereofis administered orally, for 28 days in a 28-day cycle. In otherembodiments, the compound is administered with a drug holiday. Forexample, a compound disclosed herein or a pharmaceutically acceptablesalt thereof is orally administered, e.g., for 21 days of a 28-day cyclewith a 7-day drug holiday per cycle.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said single dose ranges from about 100mg to about 1600 mg.

In any method or formulation (e.g., an oral dosage form) describedherein, a single dose of a compound disclosed herein or apharmaceutically acceptable salt thereof is 100, 200, 400, 800 or 1600mg.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said therapeutically effective amount isa single 800 mg dose, wherein said single dose provides a mean AUC(0-12)bioequivalent to a mean AUC(0-12) of from about 7798 ng*hr/ml to about9441 ng*hr/ml.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said therapeutically effective amount isa single 1600 mg dose, wherein said single dose provides a meanAUC(0-12) bioequivalent to a mean AUC(0-12) of from about 15596 ng*hr/mlto about 18882 ng*hr/ml.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said therapeutically effective amount isa single 800 mg dose, wherein said single dose provides a mean Cmaxbioequivalent to a mean Cmax of from about 1730 ng/ml to about 2063ng/ml.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said therapeutically effective amount isa single 1600 mg dose, wherein said single dose provides a mean Cmaxbioequivalent to a mean Cmax of from about 3460 ng/ml to about 4125ng/ml.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said administering comprisesadministering orally a dosage form to the subject, twice per day orthree times per day.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said single dose provides a median Tmaxof from about 1 hour to about 2 hours.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said oral dosage form or formulationcomprises an amount of therapeutic agent equivalent to about 25 mg toabout 200 mg of EPZ-6438 per unit dose.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said oral dosage form or formulationprovides an dissolution rate of at least about 90%, or at least about80%, or at least about 70% in dissolution medium (pH1.2, 37±0.5° C.)within 60 minutes from the onset of dissolution study using theApparatus 2 (Paddle Apparatus, paddle speed; 50 rpm) according to theprocedure for immediate-release dosage form in 6.10 Dissolution test ofJP16 or <711> Dissolution of USP37.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said oral dosage form or formulationprovides an dissolution rate of at least about 90%, or at least about80%, or at least about 70% in dissolution medium (pH1.2, 37±0.5° C.)within 45 minutes from the onset of dissolution study using theApparatus 2 (Paddle Apparatus, paddle speed; 50 rpm) according to theprocedure for immediate-release dosage form in 6.10 Dissolution test ofJP16 or <711> Dissolution of USP37.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said oral dosage form or formulationprovides an dissolution rate of at least about 90%, or at least about80%, or at least about 70% in dissolution medium (pH1.2, 37±0.5° C.)within 30 minutes from the onset of dissolution study using theApparatus 2 (Paddle Apparatus, paddle speed; 50 rpm) according to theprocedure for immediate-release dosage form in 6.10 Dissolution test ofJP16 or <711> Dissolution of USP37.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said oral dosage form or formulationprovides an dissolution rate of at least about 80%, or at least about75%, or at least about 70%, or at least about 60% in dissolution medium(pH4.5 acetate buffer, 37±0.5° C.) within 60 minutes from the onset ofdissolution study using the Apparatus 2 (Paddle Apparatus, paddle speed;50 rpm) according to the procedure for immediate-release dosage form in6.10 Dissolution test of JP16 or <711> Dissolution of USP37.

In any method or formulation (e.g., an oral dosage form) describedherein, in certain embodiments, said oral dosage form or formulationcomprises sodium starch glycolate or carmellose or a combination thereofas pharmaceutically acceptable carrier or excipient.

Other compounds suitable for the methods of the disclosure are describedin U.S. Publication 20120264734, the contents of which are herebyincorporated by reference in their entireties. Further, Compound (A) issuitable for administration as part of a combination therapy with one ormore other therapeutic agents or treatment modality, suitable to beadministered together, sequentially, or in alternation.

In one embodiment, Compound (A) or a pharmaceutically acceptable saltthereof is administered to the subject at a dose of approximately 100 mgto approximately 3200 mg daily, such as about 100 mg BID to about 1600mg BID (e.g., 100 mg BID, 200 mg BID, 400 mg BID, 800 mg BID, or 1600 mgBID), for treating INI1-negative tumor (e.g., rhabdoid tumor of thekidney (RTK), atypical teratoid/rhabdoid tumor (ATRT), epithelioidmalignant peripheral nerve sheath tumor, myoepithelial carcinoma, andrenal medullary carcinoma).

The use of the articles “a”, “an”, and “the” herein are to be construedto cover both the singular and the plural, unless otherwise indicatedherein or clearly contradicted by context. For example, the term “adisintegrant” refers to one or more disintegrants included in orsuitable for use in the formulation described herein. Similarly, theterm “a therapeutic agent” refers to one or more therapeutic agentsincluded in or suitable for use in the formulation described herein. Forexample, the formulation described herein can include Compound (A) aloneas the therapeutic agent or active ingredient or include a mixture ofCompound (A) and another compound (e.g., HBr salt of Compound (A) oranother anti-cancer drug). The terms “comprising”, “having”,“including”, and “containing” are to be construed as open terms (i.e.,meaning “including but not limited to”) unless otherwise noted.Additionally whenever “comprising” or another open-ended term is used inan embodiment, it is to be understood that the same embodiment can bemore narrowly claimed using the intermediate term “consistingessentially of” or the closed term “consisting of.”

The concentration of the therapeutic agent in the formulation isexpressed as equivalent to a certain amount of Compound (A). As usedherein, the term “equivalent” amount or weight percentage refers to thequantity of the drug substance that is adjusted as per potencyadjustment factor, a value derived for the assay value obtained fromCompound (A). Methods for determining the equivalent amount are wellknown in the art (see, e.g., http://www.fda.gov/downloads/Drugs/ . . ./Guidances/ucm070246.pdf).

The term “about”, “approximately”, or “approximate”, when used inconnection with a numerical value, means that a collection or ranger ofvalues is included. For example, “about ×” includes a range of valuesthat are ±10%, +5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X, where X is anumerical value. In addition, “about X” may also include a range ofX±0.5, X±0.4, X±0.3, X±0.2, or X±0.1, where X is a numerical value. Inone embodiment, the term “about” refers to a range of values which are5% more or less than the specified value. In another embodiment, theterm “about” refers to a range of values which are 2% more or less thanthe specified value. In another embodiment, the term “about” refers to arange of values which are 1% more or less than the specified value.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent disclosure includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In addition, a crystal polymorphism may bepresent for the compounds represented by the formula. It is noted thatany crystal form, crystal form mixture, or anhydride or hydrate thereofis included in the scope of the present disclosure. Furthermore,so-called metabolite which is produced by degradation of the presentcompound in vivo is included in the scope of the present disclosure.

Furthermore, the structures and other compounds discussed in thisdisclosure include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

The term “crystal polymorphs”, “polymorphs” or “crystalline forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different XRPD patterns, infrared spectral, melting points, densityhardness, crystal shape, optical and electrical properties, stabilityand solubility. Recrystallization solvent, rate of crystallization,storage temperature, and other factors may cause one crystal form todominate. Crystal polymorphs of the compounds can be prepared bycrystallization under different conditions.

Compounds of the disclosure may be crystalline, semi-crystalline,non-crystalline, amorphous, mesomorphous, etc.

Additionally, the compounds or crystalline forms of the presentdisclosure, for example, the salts of the compounds or crystallineforms, can exist in either hydrated or unhydrated (the anhydrous) formor as solvates with other solvent molecules. Nonlimiting examples ofhydrates include hemihydrates, monohydrates, dihydrates, trihydrates,etc. Nonlimiting examples of solvates include ethanol solvates, acetonesolvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non-stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O. A hemihydrate isformed by the combination of one molecule of water with more than onemolecule of the substance in which the water retains its molecular stateas H₂O.

The present disclosure is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

As used herein, a “subject” is interchangeable with a “subject in needthereof”, both of which refer to a subject having a disorder in whichEZH2-mediated protein methylation plays a part, or a subject having anincreased risk of developing such disorder relative to the population atlarge. A “subject” includes a mammal. The mammal can be e.g., a human orappropriate non-human mammal, such as primate, mouse, rat, dog, cat,cow, horse, goat, camel, sheep or a pig. The subject can also be a birdor fowl. In one embodiment, the mammal is a human. A subject in needthereof can be one who has been previously diagnosed or identified ashaving cancer or a precancerous condition. A subject in need thereof canalso be one who has (e.g., is suffering from) cancer or a precancerouscondition. Alternatively, a subject in need thereof can be one who hasan increased risk of developing such disorder relative to the populationat large (i.e., a subject who is predisposed to developing such disorderrelative to the population at large). A subject in need thereof can havea precancerous condition. A subject in need thereof has an INI1-negativetumor.

INI1 is a regulatory complex that opposes the enzymatic function ofEZH2. Due to a variety of genetic alterations, INI1 loses its regulatoryfunction. As a result, EZH2 activity is misregulated, causing EZH2 toplay a driving, oncogenic role in a set of genetically defined cancersthat include rhabdoid tumor of the kidney (RTK), atypicalteratoid/rhabdoid tumor (ATRT), epithelioid malignant peripheral nervesheath tumor, myoepithelial carcinoma, and renal medullary carcinoma.

A subject in need thereof can have refractory or resistant cancer (i.e.,cancer that doesn't respond or hasn't yet responded to treatment). Thesubject may be resistant at start of treatment or may become resistantduring treatment. In some embodiments, the subject in need thereof hascancer recurrence following remission on most recent therapy. In someembodiments, the subject in need thereof received and failed all knowneffective therapies for cancer treatment. In some embodiments, thesubject in need thereof received at least one prior therapy. In apreferred embodiment, the subject has cancer or a cancerous condition.

In certain embodiments, in any method described herein, the subject isan adult patient aged 18 years or older.

In certain embodiments, in any method described herein, the subject is apediatric patient aged 12 months or younger (e.g., between 3 and 12months old).

In certain embodiments, in any method described herein, the subject is asubject older than 12 months but younger than 18 years old.

In any method described herein, the subject can be a pediatric(non-adult) patient aged 3 months to 18 years.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentdisclosure, or a pharmaceutically acceptable salt, prodrug, metabolite,polymorph or solvate thereof, to alleviate the symptoms or complicationsof a disease, condition or disorder, or to eliminate the disease,condition or disorder. The term “treat” can also include treatment of acell in vitro or an animal model.

A compound of the present disclosure, or a pharmaceutically acceptablesalt, prodrug, metabolite, polymorph or solvate thereof, can or may alsobe used to prevent a relevant disease, condition or disorder, or used toidentify suitable candidates for such purposes. As used herein,“preventing,” “prevent,” or “protecting against” describes reducing oreliminating the onset of the symptoms or complications of such disease,condition or disorder.

The methods and uses described herein may include steps of detecting thepresence or absence of one or more EZH2 mutations in a sample from asubject in need thereof prior to and/or after the administration of acompound or composition described herein to the subject. By “sample” itmeans any biological sample derived from the subject, includes but isnot limited to, cells, tissues samples, body fluids (including, but notlimited to, mucus, blood, plasma, serum, urine, saliva, and semen),tumor cells, and tumor tissues. Preferably, the sample is selected frombone marrow, peripheral blood cells, blood, plasma and serum. Samplescan be provided by the subject under treatment or testing. Alternativelysamples can be obtained by the physician according to routine practicein the art.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the disclosure.

The present disclosure also provides pharmaceutical compositionscomprising one or more active compounds (e.g., Compound (A) or a saltthereof) in combination with at least one pharmaceutically acceptableexcipient or carrier.

In one embodiment, the pharmaceutical composition is in bulk or in unitdosage form. The term “unit dosage form” as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The unit dosageform is any of a variety of forms, including, for example, an IV bag, atablet, a single pump on an aerosol inhaler or a vial. The quantity ofactive ingredient (e.g., a formulation of the disclosed compound orsalt, hydrate, solvate or isomer thereof) in a unit dose of compositionis an effective amount and is varied according to the particulartreatment involved. One skilled in the art will appreciate that it issometimes necessary to make routine variations to the dosage dependingon the age and condition of the patient. The dosage will also depend onthe route of administration. A variety of routes are contemplated,including oral, pulmonary, rectal, parenteral, transdermal,subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational,buccal, sublingual, intrapleural, intrathecal, intranasal, and the like.Dosage forms for the topical or transdermal administration of a compoundof this disclosure include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches and inhalants. In one embodiment, theactive compound is mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants that are required.

In one embodiment, the unit dosage form is an oral dosage form. In oneembodiment, the unit dosage form is a tablet. In one embodiment, theunit dosage form is an oral suspension. In one embodiment, the unitdosage form is an oral suspension and the subject is a pediatricsubject.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient. For example, a pharmaceutically acceptable excipientused for the formulation of the disclosure can be a diluent or inertcarrier, a lubricant, a binder, or a combination thereof. Thepharmaceutically acceptable excipient used for the formulation of thedisclosure can further include a filler, an anti-microbial agent, anantioxidant, an anti-caking agent, a coating agent, or a mixturethereof.

Examples of pharmaceutically acceptable excipients include, but are notlimited to binders, fillers, disintegrants, lubricants, anti-microbialagents, antioxidant, and coating agents.

Exemplary binders include, but are not limited to corn starch, potatostarch, other starches, gelatin, natural and synthetic gums such asacacia, xanthan, sodium alginate, alginic acid, other alginates,powdered tragacanth, guar gum, cellulose and its derivatives (e.g.,ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium,sodium carboxymethyl cellulose), polyvinyl pyrrolidone (e.g., povidone,crospovidone, copovidone, etc.), methyl cellulose, Methocel,pre-gelatinized starch (e.g., STARCH 1500® and STARCH 1500 LM®, sold byColorcon, Ltd.), hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose (FMC Corporation, Marcus Hook,Pa., USA), Emdex, Plasdone, or mixtures thereof, FILLERS: talc, calciumcarbonate (e.g., granules or powder), dibasic calcium phosphate,tribasic calcium phosphate, calcium sulfate (e.g., granules or powder),microcrystalline cellulose, powdered cellulose, dextrates, kaolin,mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch,dextrose, fructose, honey, lactose anhydrate, lactose monohydrate,lactose and aspartame, lactose and cellulose, lactose andmicrocrystalline cellulose, maltodextrin, maltose, mannitol,microcrystalline cellulose &amp; guar gum, molasses, sucrose, ormixtures thereof.

Exemplary disintegrants include, but are not limited to: agar-agar,alginic acid, calcium carbonate, microcrystalline cellulose,croscarmellose sodium, crospovidone, polacrilin potassium, sodium starchglycolate (such as Explotab), potato or tapioca starch, other starches,pre-gelatinized starch, clays, other algins, other celluloses, gums(like gellan), low-substituted hydroxypropyl cellulose, ployplasdone, ormixtures thereof.

Exemplary lubricants include, but are not limited to: calcium stearate,magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol,mannitol, polyethylene glycol, other glycols, compritol, stearic acid,sodium lauryl sulfate, sodium stearyl fumarate (such as Pruv), vegetablebased fatty acids lubricant, talc, hydrogenated vegetable oil (e.g.,peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, cornoil and soybean oil), zinc stearate, ethyl oleate, ethyl laurate, agar,syloid silica gel (AEROSIL 200, W.R. Grace Co., Baltimore, Md. USA), acoagulated aerosol of synthetic silica (Degussa Corp., Plano, Tex. USA),a pyrogenic silicon dioxide (CAB-O-SIL, Cabot Co., Boston, Mass. USA),or mixtures thereof.

Exemplary anti-caking agents include, but are not limited to: calciumsilicate, magnesium silicate, silicon dioxide, colloidal silicondioxide, talc, or mixtures thereof.

Exemplary antimicrobial agents include, but are not limited to:benzalkonium chloride, benzethonium chloride, benzoic acid, benzylalcohol, butyl paraben, cetylpyridinium chloride, cresol, chlorobutanol,dehydroacetic acid, ethylparaben, methylparaben, phenol, phenylethylalcohol, phenoxyethanol, phenylmercuric acetate, phenylmercuric nitrate,potassium sorbate, propylparaben, sodium benzoate, sodiumdehydroacetate, sodium propionate, sorbic acid, thimersol, thymo, ormixtures thereof.

Exemplary antioxidants include, but are not limited to: ascorbic acid,BHA, BHT, EDTA, or mixture thereof.

Exemplary coating agents include, but are not limited to: sodiumcarboxymethyl cellulose, cellulose acetate phthalate, ethylcellulose,gelatin, pharmaceutical glaze, hydroxypropyl cellulose, hydroxypropylmethylcellulose (hypromellose), hydroxypropyl methyl cellulosephthalate, methylcellulose, polyethylene glycol, polyvinyl acetatephthalate, shellac, sucrose, titanium dioxide, carnauba wax,microcrystalline wax, gellan gum, maltodextrin, methacrylates,microcrystalline cellulose and carrageenan or mixtures thereof.

The formulation described herein can also include other excipients andcategories thereof including but not limited to Pluronic®, Poloxamers(such as Lutrol® and Poloxamer 188), ascorbic acid, glutathione,protease inhibitors (e.g. soybean trypsin inhibitor, organic acids), pHlowering agents, creams and lotions (like maltodextrin andcarrageenans); materials for chewable tablets (like dextrose, fructose,lactose monohydrate, lactose and aspartame, lactose and cellulose,maltodextrin, maltose, mannitol, microcrystalline cellulose and guargum, sorbitol crystalline); parenterals (like mannitol and povidone);plasticizers (like dibutyl sebacate, plasticizers for coatings,polyvinylacetate phthalate); powder lubricants (like glyceryl behenate);spheres for coating (like sugar spheres); spheronization agents (likeglyceryl behenate and microcrystalline cellulose); suspending/gellingagents (like carrageenan, gellan gum, mannitol, microcrystallinecellulose, povidone, sodium starch glycolate, xanthan gum); sweeteners(like aspartame, aspartame and lactose, dextrose, fructose, honey,maltodextrin, maltose, mannitol, molasses, sorbitol crystalline,sorbitol special solution, sucrose); wet granulation agents (likecalcium carbonate, lactose anhydrous, lactose monohydrate, maltodextrin,mannitol, microcrystalline cellulose, povidone, starch), caramel,carboxymethylcellulose sodium, cherry cream flavor and cherry flavor,citric acid anhydrous, citric acid, confectioner's sugar, D&C Red No.33, D&C Yellow #10 Aluminum Lake, disodium edetate, ethyl alcohol 15%,FD&C Yellow No. 6 aluminum lake, FD&C Blue #1 Aluminum Lake, FD&C BlueNo. 1, FD&C blue no. 2 aluminum lake, FD&C Green No. 3, FD&C Red No. 40,FD&C Yellow No. 6 Aluminum Lake, FD&C Yellow No. 6, FD&C Yellow No. 10,glycerol palmitostearate, glyceryl monostearate, indigo carmine,lecithin, mannitol, methyl and propyl parabens, mono ammoniumglycyrrhizinate, natural and artificial orange flavor, pharmaceuticalglaze, poloxamer 188, Polydextrose, polysorbate 20, polysorbate 80,polyvidone, pregelatinized corn starch, pregelatinized starch, red ironoxide, saccharin sodium, sodium carboxymethyl ether, sodium chloride,sodium citrate, sodium phosphate, strawberry flavor, synthetic blackiron oxide, synthetic red iron oxide, titanium dioxide, and white wax.

In certain embodiments, the formulation of the disclosure is a solidoral dosage form that may optionally be treated with coating systems(e.g. Opadry® fx film coating system) to be coated with for exampleOpadry® blue (OY-LS-20921), Opadry® white (YS-2-7063), Opadry® white(YS-1-7040), and black ink (S-1-8 106).

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic selected for administration.Therapeutically effective amounts for a given situation can bedetermined by routine experimentation that is within the skill andjudgment of the clinician.

The pharmaceutical compositions of the present disclosure containingactive compounds may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol and sorbitol, and sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms disclosed herein are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the EZH2 inhibitor compoundsdescribed herein, or the pharmaceutical compositions used in accordancewith the disclosure vary depending on the agent, the age, weight, andclinical condition of the recipient patient, and the experience andjudgment of the clinician or practitioner administering the therapy,among other factors affecting the selected dosage. Generally, the doseshould be sufficient to result in slowing, and preferably regressing,the growth of the tumors and also preferably causing complete regressionof the cancer. Dosages can range from about 0.01 mg/kg per day to about5000 mg/kg per day. In preferred aspects, dosages can range from about 1mg/kg per day to about 1000 mg/kg per day. In an aspect, the dose willbe in the range of about 0.1 mg/day to about 50 g/day; about 0.1 mg/dayto about 25 g/day; about 0.1 mg/day to about 10 g/day; about 0.1 mg toabout 3 g/day; or about 0.1 mg to about 1 g/day, in single, divided, orcontinuous doses (which dose may be adjusted for the patient's weight inkg, body surface area in m², and age in years). An effective amount of apharmaceutical agent is that which provides an objectively identifiableimprovement as noted by the clinician or other qualified observer. Forexample, regression of a tumor in a patient may be measured withreference to the diameter of a tumor. Decrease in the diameter of atumor indicates regression. Regression is also indicated by failure oftumors to reoccur after treatment has stopped. As used herein, the term“dosage effective manner” refers to amount of an active compound toproduce the desired biological effect in a subject or cell.

It is especially advantageous to formulate oral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe disclosure are dictated by and directly dependent on the uniquecharacteristics of the active Compound (A) and the particulartherapeutic effect to be achieved.

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the disclosure vary depending onthe agent, the age, weight, and clinical condition of the recipientpatient, and the experience and judgment of the clinician orpractitioner administering the therapy, among other factors affectingthe selected dosage. Generally, the dose should be sufficient to resultin slowing, and preferably regressing, the growth of the tumors and alsopreferably causing complete regression of the cancer. An effectiveamount of a pharmaceutical agent is that which provides an objectivelyidentifiable improvement as noted by the clinician or other qualifiedobserver. For example, regression of a tumor in a patient may bemeasured with reference to the diameter of a tumor. Decrease in thediameter of a tumor indicates regression. Regression is also indicatedby failure of tumors to reoccur after treatment has stopped. As usedherein, the term “dosage effective manner” refers to amount of an activecompound to produce the desired biological effect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds in the formulation of the present disclosure are capableof further forming salts. All of these forms are also contemplatedwithin the scope of the claimed disclosure.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present disclosure wherein the parent compoundis modified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present disclosure alsoencompasses salts formed when an acidic proton present in the parentcompound either is replaced by a metal ion, e.g., an alkali metal ion,an alkaline earth ion, or an aluminum ion; or coordinates with anorganic base such as ethanolamine, diethanolamine, triethanolamine,tromethamine, N-methylglucamine, and the like. In the salt form, it isunderstood that the ratio of the compound to the cation or anion of thesalt can be 1:1, or any ration other than 1:1, e.g., 3:1, 2:1, 1:2, or1:3.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) or crystalforms (polymorphs) as defined herein, of the same salt.

The compounds, or pharmaceutically acceptable salts, esters or prodrugsthereof, are administered orally, nasally, transdermally, pulmonary,inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the disclosure can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

Cancer is a group of diseases that may cause almost any sign or symptom.The signs and symptoms will depend on where the cancer is, the size ofthe cancer, and how much it affects the nearby organs or structures. Ifa cancer spreads (metastasizes), then symptoms may appear in differentparts of the body.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound disclosed herein, or apharmaceutically acceptable salt, solvate, analog or derivative thereof.Preferably, the average survival time is increased by more than 30 days;more preferably, by more than 60 days; more preferably, by more than 90days; and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.An increase in average survival time of a population may be measured,for example, by calculating for a population the average length ofsurvival following initiation of treatment with an active compound. Anincrease in average survival time of a population may also be measured,for example, by calculating for a population the average length ofsurvival following completion of a first round of treatment with anactive compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound disclosedherein, or a pharmaceutically acceptable salt, solvate, analog orderivative thereof. Preferably, the mortality rate is decreased by morethan 2%; more preferably, by more than 5%; more preferably, by more than10%; and most preferably, by more than 25%. A decrease in the mortalityrate of a population of treated subjects may be measured by anyreproducible means. A decrease in the mortality rate of a population maybe measured, for example, by calculating for a population the averagenumber of disease-related deaths per unit time following initiation oftreatment with an active compound. A decrease in the mortality rate of apopulation may also be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing completion of a first round of treatment with an activecompound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at least5%; more preferably, by at least 10%; more preferably, by at least 20%;more preferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.The size of an area or zone of cellular proliferation may be measured asa diameter or width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compounddisclosed herein, or a pharmaceutically acceptable salt or solvatethereof, acts selectively on a cancer or precancerous cell but not on anormal cell. Preferably, a compound disclosed herein, or apharmaceutically acceptable salt or solvate thereof, acts selectively tomodulate one molecular target (e.g., a target protein methyltransferase)but does not significantly modulate another molecular target (e.g., anon-target protein methyltransferase). The disclosure also provides amethod for selectively inhibiting the activity of an enzyme, such as aprotein methyltransferase. Preferably, an event occurs selectively inpopulation A relative to population B if it occurs greater than twotimes more frequently in population A as compared to population B. Anevent occurs selectively if it occurs greater than five times morefrequently in population A. An event occurs selectively if it occursgreater than ten times more frequently in population A; more preferably,greater than fifty times; even more preferably, greater than 100 times;and most preferably, greater than 1000 times more frequently inpopulation A as compared to population B. For example, cell death wouldbe said to occur selectively in cancer cells if it occurred greater thantwice as frequently in cancer cells as compared to normal cells.

A composition disclosed herein does not significantly modulate theactivity of a molecular target if the addition of the compound does notstimulate or inhibit the activity of the molecular target by greaterthan 10% relative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a protein methyltransferase isozyme alpha in comparisonto a protein methyltransferase isozyme beta). Preferably, a compounddisclosed herein, or a pharmaceutically acceptable salt or solvatethereof, demonstrates a minimum of a fourfold differential, preferably atenfold differential, more preferably a fifty fold differential, in thedosage required to achieve a biological effect. Preferably, a compounddisclosed herein, or a pharmaceutically acceptable salt or solvatethereof, demonstrates this differential across the range of inhibition,and the differential is exemplified at the IC₅₀, i.e., a 50% inhibition,for a molecular target of interest.

Administering a composition disclosed herein to a cell or a subject inneed thereof can result in modulation (i.e., stimulation or inhibition)of an activity of a protein methyltransferase of interest.

Administering a compound disclosed herein, e.g., a compositioncomprising any compound disclosed herein or pharmaceutically acceptablesalt thereof, and one or more other therapeutic agents, such asprednisone, to a cell or a subject in need thereof results in modulation(i.e., stimulation or inhibition) of an activity of an intracellulartarget (e.g., substrate). Several intracellular targets can be modulatedwith the compounds disclosed herein, including, but not limited to,protein methyltrasferase.

Activating refers to placing a composition of matter (e.g., protein ornucleic acid) in a state suitable for carrying out a desired biologicalfunction. A composition of matter capable of being activated also has anunactivated state. An activated composition of matter may have aninhibitory or stimulatory biological function, or both.

Elevation refers to an increase in a desired biological activity of acomposition of matter (e.g., a protein or a nucleic acid). Elevation mayoccur through an increase in concentration of a composition of matter.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc Natl Acad Sci USA. 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

Preferably, an effective amount of a composition disclosed herein, or apharmaceutically acceptable salt or solvate thereof, is notsignificantly cytotoxic to normal cells. A therapeutically effectiveamount of a compound is not significantly cytotoxic to normal cells ifadministration of the compound in a therapeutically effective amountdoes not induce cell death in greater than 10% of normal cells. Atherapeutically effective amount of a compound does not significantlyaffect the viability of normal cells if administration of the compoundin a therapeutically effective amount does not induce cell death ingreater than 10% of normal cells. In an aspect, cell death occurs byapoptosis.

Contacting a cell with a composition disclosed herein, or apharmaceutically acceptable salt or solvate thereof, can induce oractivate cell death selectively in cancer cells. Administering to asubject in need thereof a compound disclosed herein, or apharmaceutically acceptable salt or solvate thereof, can induce oractivate cell death selectively in cancer cells. Contacting a cell witha composition disclosed herein, or a pharmaceutically acceptable salt orsolvate thereof, can induce cell death selectively in one or more cellsaffected by a cell proliferative disorder. Preferably, administering toa subject in need thereof a composition disclosed herein, or apharmaceutically acceptable salt or solvate thereof, induces cell deathselectively in one or more cells affected by a cell proliferativedisorder.

The present disclosure relates to a method of treating or preventingcancer by administering a composition disclosed herein, or apharmaceutically acceptable salt or solvate thereof, to a subject inneed thereof, where administration of the composition disclosed herein,or a pharmaceutically acceptable salt or solvate thereof, results in oneor more of the following: prevention of cancer cell proliferation byaccumulation of cells in one or more phases of the cell cycle (e.g. G1,G1/S, G2/M), or induction of cell senescence, or promotion of tumor celldifferentiation; promotion of cell death in cancer cells viacytotoxicity, necrosis or apoptosis, without a significant amount ofcell death in normal cells, antitumor activity in animals with atherapeutic index of at least 2. As used herein, “therapeutic index” isthe maximum tolerated dose divided by the efficacious dose.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present disclosure areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentdisclosure. The examples do not limit the claimed disclosure. Based onthe present disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present disclosure.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless otherwise clear from the context, all numerical values providedherein are modified by the term “about.”

EXAMPLE Example 1

INI1 is a critical component of the SWI/SNF regulatory complex, achromatin remodeler that acts in opposition to EZH2. INI1-negativetumors have altered SWI/SNF function, resulting in aberrant andoncogenic EZH2 activity. This activity can be targeted by small moleculeinhibitors of EZH2 such as tazemetostat. INI1-negative tumors aregenerally aggressive and are poorly served by current treatments. Forexample, current treatment of MRT, a well-studied INI1-negative tumor,consists of surgery, chemotherapy and radiation therapy, which areassociated with limited efficacy and significant treatment-relatedmorbidity.

The adult phase 2 multicenter study will enroll up to 90 patients inthree cohorts. The first cohort will be comprised of patients withmalignant rhabdoid tumor (MRT), rhabdoid tumor of the kidney (RTK) andatypical teratoid/rhabdoid tumor (ATRT). The second cohort will becomprised of patients with other INI1-negative tumors includingepitheloid sarcoma, epithelioid malignant peripheral nerve sheath tumor,extraskeletal myxoid chondrosarcoma, myoepithelial carcinoma, and renalmedullary carcinoma. The third cohort will be comprised of patients withsynovial sarcoma. Dosing in all three cohorts will be at the recommendedphase 2 dose of 800 mg twice per day (BID) with a tablet formulation.The primary endpoint is overall response rate (ORR) for patients withINI1-negative tumors and progression-free survival (PFS) for patientswith synovial sarcoma. Secondary endpoints include duration of response,overall survival (OS), PFS for patients with INI1-negative tumors,safety and pharmacokinetics (PK).

The pediatric phase 1 multicenter study will enroll approximately 40patients in a dose escalation design, followed by dose expansion, withan oral suspension of tazemetostat. The study will enroll subjects withINI1-negative tumors or synovial sarcoma. INI1-negative tumors includeMRT, ATRT, RTK, and other INI1-negative tumors as previously described.The primary endpoint of study is safety with the objective ofestablishing the recommended phase 2 dose in pediatric patients.Secondary endpoints include PK, ORR, duration of response, PFS and OS.

The disclosure can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the disclosure described herein. Scope of thedisclosure is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A method for treating an INI1-negative tumor comprising administeringa therapeutically effective amount of an EZH2 inhibitor to a subject inneed thereof, wherein the INI1-negative tumor is selected fromepithelioid malignant peripheral nerve sheath tumor and myoepithelialcarcinoma, wherein the subject is younger than 18 years old and whereinthe EZH2 inhibitor is administered to the subject at a dose of about 100mg to about 3200 mg daily. 2.-3. (canceled)
 4. The method of claim 1,wherein the INI11-negative tumor is epithelioid malignant peripheralnerve sheath tumor.
 5. The method of claim 1, wherein the INI11-negativetumor is myoepithelial carcinoma.
 6. (canceled)
 7. The method of claim1, wherein the EZH2 inhibitor is administered orally.
 8. The method ofclaim 1, wherein the subject is a human being.
 9. (canceled)
 10. Themethod of claim 1, wherein the EZH2 inhibitor is Compound (A), havingthe following formula:

or a pharmaceutically acceptable salt thereof.
 11. (canceled)
 12. Themethod of claim 1, wherein the EZH2 inhibitor is administered to thesubject at a dose of about 100 mg BID to about 1600 mg BID.
 13. Themethod of claim 1, wherein the EZH2 inhibitor is administered to thesubject at a dose of about 100 mg BID, 200 mg BID, 400 mg BID, 800 mgBID, or about 1600 mg BID.
 14. The method of claim 13, wherein the EZH2inhibitor is administered to the subject at a dose of 800 mg BID. 15.The method of claim 1, wherein the EZH2 inhibitor is selected from thegroup consisting of

and pharmaceutically acceptable salts thereof.
 16. The method of claim15, wherein the EZH2 inhibitor is:

or a pharmaceutically acceptable salt thereof.